The CDKN2 gene has been recently localized to a chromosomal region found to be deleted in leukemias and solid tumors. CDKN2 encodes a 16 kDa protein product (p16INK4A), which functions as a specific inhibitor or the cyclin-dependent kinases 4 and 6. There have been many reports indicating a higher frequency of deletions of the CDKN2 gene in a variety of tumor cell lines, in comparison to primary tumors. These studies raise the possibility that deletions of CDKN2 may be a rare event in primary tumors, and in fact arise in vitro, during the establishment of permanent cell lines. To address this issue, we determined whether the CDKN2 gene deletions found in acute lymphoblastic leukemia (ALL) cell lines are also detected in the primary leukemia samples. Eleven cell lines were identified which had available frozen primary samples of their original leukemic tissue. Five out of 11 of these cell lines, as well as their primary samples had homozygous CDKN2 deletions. The remaining six cell lines and their primary samples retained at least one copy of the CDKN2 gene. Of the six CDKN2+ cell lines, five expressed CDKN2 mRNA, but only one of these expressed the p16 protein product (as did its primary sample). Our results indicate that CDKN2 deletions present in the studied ALL cell lines arose in the primary leukemic cells, and not during cell line establishment or prolonged in vitro culture.